Chronic hypertension is typically associated with increased peripheral vascular resistance. This is due, in part, to enhanced arterial smooth muscle contractility, which is regulated by cellular Ca 2[unreadable]. Recent findings suggest that Ca 2[unreadable] influx through TRPC-encoded store-operated channels (SOCs) in the plasma membrane (PM) may regulate myogenic tone. This project tests the hypothesis that TRPC-encoded SOCs are localized to PM microdomains adjacent to underlying junctional sarcoplasmic reticulum (jSR) and are functionally linked to distinct SR Ca 2+ stores [(IP3)/cyclopiazonic acid (CPA)- and caffeine/ryanodine (CAF/RY)-sensitive)]. I propose that the altered Ca 2[unreadable] homeostasis in arterial smooth muscle cells (ASMCs) from ouabain hypertensive (OH)rats is duel in part, to upregulated expression of TRPC-encoded SOCs. Changes in TRPC expression under conditions of o_2Na + pump inhibition may result from elevated SR Ca 2[unreadable] ([Ca 2 ]sR). The Specific Aims address the following questions: 1. Which TRPC proteins (TRPC1-7) are involved in Ca 2+ entry following unloading of distinct SR stores in freshly isolated ASMCs? Specific TRPC proteins will be downregulated by antisense oligos for TRPC genes to identify those associated with CPA- and/or CAF/RY-induced store-operated Ca 2[unreadable] entry (SOCE). The latter will be probed by measuring SOCE- evoked changes in cytosolic Ca 2[unreadable]concentration ([Ca2+]c_) using fluorescence microscopy. TRPC expression in ASMCs will be identified by PCR and immunoblotting. 2. Is Ca 2+ entry through TRPC-encoded SOCs localized to PM microdomains adjacent to underlying jSR in ASMCs? a) High resolution imaging with near- membrane Ca 2+indicators will be used to localize the sites of SOCE;b) Immunocytochemistry on the same cell will determine the location of TRPC proteins within PM microdomains. 3. Which Ca 2[unreadable] transport and storage mechanisms are altered in ASMCs from OH rats? Ca 2[unreadable] transport proteins will be quantified and compared with resting [Ca2+]_, [Ca2*]sa and SOCE in ASMCs from control and OH rats. 4. Does nanomolar ouabain affect expression of TRPC-encoded SOCs in ASMCs in vitro, and what is the role of [Ca2+]sR? Mice with genetically altered Na + pumps and Na/Ca exchanger will be used. The results of this project will likely lead to new insights into the role of localized Ca 2+ transport mechanisms in the regulation of vascular tone and may suggest innovative approaches for the development of novel therapeutics for vascular dysfunction. i